Validation of a new LC-MS/MS method for determination of Δ9-tetrahydrocannabinol (THC) and 11-nor-9-carboxy-Δ9-tetrahydrocannabinol (THC-COOH) in hair and its application to a preliminary study on relationship between their concentrations and age, gender, consumption habits and hair features. After washing and cutting steps, a 25-mg aliquot of hair was hydrolysed (1 mL of 1 M NaOH at 65 °C, 20 min), acidified (1.5 mL 1 M of H2SO4) and then liquid-liquid extracted with hexane/ethyl acetate (90/10, v/v). The organic phase was dried and reconstituted with 100 μL of mobile phase (5 mM aqueous formic acid and acetonitrile 50:50). Chromatographic separation was performed through a C18 column. Acquisition was in multiple reaction monitoring for the following transitions: 315→259, 193 m/z, for THC; 318→196, 123 m/z, for THC-d3; 345→299, 193 m/z for THC-COOH; 348→196, 302 m/z for THC-COOH-d3. Validation was achieved following the Scientific Working Group for Forensic Toxicology's guidelines. For this study, scalp hair samples (> 3 cm length) were provided by subjects tested for THC consumption for personal purposes (i.e. workplace drug testing, personal use proving). A short questionnaire was also administrated in order to collect information about age, gender, consumption habits, hair treatment. Correlation between THC and THC-COOH hair concentrations was analysed by the Spearman's rank correlation coefficient. In order to study the influences of the several variables, we introduced a new value, Sqrt(THC*THCCOOH). The effectiveness and reliability of it were proved by the Principal Component Analysis. Relationships between the Sqrt(THC*THCCOOH) and the variables were studied through the Stepwise regression ( P = 0.05). Normality of data distribution was tested by Shapiro-Wilk test. The Lower limits of quantification were 10.0 (THC) and 0.2 (THC-COOH) pg/mg. Accuracy and precision always met the acceptable criteria. Recoveries were > 75% and ion suppression were observed for both the compounds. 126 hair samples were analysed and 54 (42.9%) were positive both for THC and THC-COOH; none of samples was positive for a single substance. Concentrations ranged from 0.18 to 1.75 ng/mg (median: 0.78 ng/mg) for THC and from 0.04 to 0.85 ng/mg (median: 0.31 ng/mg) for THC-COOH. Both compounds were always above the cut-off levels suggested by SoHT and EWDTS at 50 and 0.2 pg/mg, respectively. Statistically significant differences were observed among the age groups, consumption habits and cosmetic treatments. THC and THC-COOH decreased with age as previously observed by Han et al., even if differences were not statistically significant (Han, Forensic Science International, 2011, 210, 201–205). A new LC-MS/MS method was validated and successfully applied to a preliminary study on the influence of age, gender, consumption habits and hair treatment and morphology.